U.S. patent application number 12/857043 was filed with the patent office on 2011-03-17 for inhibitors of hiv replication and method of treatment of hiv infections.
This patent application is currently assigned to Centre National De La Recherche Scientifique (C.N.R.S.). Invention is credited to Christian DeVaux, Gilles Divita, Roger S. Goody, Veronique Hebmann, Frederic Heitz, Jean Mery, Catherine May Morris.
Application Number | 20110064793 12/857043 |
Document ID | / |
Family ID | 24601279 |
Filed Date | 2011-03-17 |
United States Patent
Application |
20110064793 |
Kind Code |
A1 |
DeVaux; Christian ; et
al. |
March 17, 2011 |
INHIBITORS OF HIV REPLICATION AND METHOD OF TREATMENT OF HIV
INFECTIONS
Abstract
The invention is drawn to a novel class of drugs directed
against HIV, comprising a peptide or analog comprising a
decapeptide, said decapeptide containing (from N-terminus to the
C-terminus) a basic amino acid in position 1, an acidic amino acid
in positions 2 and 5, and a tryptophan in positions 4, 7, and 8,
and to a method of treatment of HIV infections, in particular
multidrug-resistant HIV infections.
Inventors: |
DeVaux; Christian;
(Montpellier, FR) ; Hebmann; Veronique; (Saint
Bauzille de Montmel, FR) ; Divita; Gilles;
(Villeurbanne, FR) ; Heitz; Frederic; (Grabels,
FR) ; Morris; Catherine May; (San Diego, CA) ;
Mery; Jean; (Saint Gely due Fesc, FR) ; Goody; Roger
S.; (Dortmund, DE) |
Assignee: |
Centre National De La Recherche
Scientifique (C.N.R.S.)
Paris
FR
|
Family ID: |
24601279 |
Appl. No.: |
12/857043 |
Filed: |
August 16, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09648557 |
Aug 25, 2000 |
7790171 |
|
|
12857043 |
|
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Current U.S.
Class: |
424/450 ;
514/3.8 |
Current CPC
Class: |
C07K 14/005 20130101;
C12N 2740/16222 20130101; A61K 38/00 20130101; A61P 31/18 20180101;
C07K 2319/00 20130101 |
Class at
Publication: |
424/450 ;
514/3.8 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61K 38/08 20060101 A61K038/08; A61P 31/18 20060101
A61P031/18 |
Claims
1. A method for treating or inhibiting an HIV infection, comprising
administering to a human in need thereof a therapeutically
effective amount of an inhibitor of HIV replication, wherein: the
inhibitor of HIV replication comprises an antiviral peptide; the
antiviral peptide consists of a decapeptide containing (from the
N-terminus to the C-terminus) a basic amino acid at position 1; an
acidic amino acid at positions 2 and 5; a tryptophan at positions
4, 7, and 8; a threonine, isoleucine or valine at position 3; a
threonine, alanine, or glutamine at position 6; a threonine,
alanine, valine, isoleucine, methionine, or aspartate at position
9; and a glutamate, aspartate or asparagine at position 10; and the
decapeptide inhibits the dimerization of HIV reverse
transcriptase.
2. The method of claim 1, wherein the basic amino acid at position
1 is lysine or arginine.
3. The method of claim 1, wherein the acidic amino acid at position
2 is glutamate.
4. The method of claim 1, wherein the amino acid at position 5 is
glutamate.
5. The method of claim 1, wherein the decapeptide is selected from
the group consisting of KETWETWWTE (SEQ ID NO:1), KETWATWWTE (SEQ
ID NO:22), and KEAWETWWTE (SEQ ID NO:23).
6. The method of claim 1, wherein the decapeptide is not KETWETWWTE
(SEQ ID NO: 1).
7. The method of claim 1, wherein the inhibitor of HIV replication
further comprises a pharmaceutically acceptable excipient.
8. The method of claim 1, wherein the inhibitor of HIV replication
further comprises a vector that allows penetration of the antiviral
peptide into a mammalian cell.
9. The method of claim 8, wherein the vector is selected from the
group consisting of a liposome, a polymeric protein-binding cation,
a protein, a peptide, a microparticle, and a nanoparticle.
10. The method of claim 9, wherein the vector is a peptide.
11. The method of claim 10, wherein the peptide is an MPG peptidyl
carrier.
12. The method of claim 11, wherein the MPG peptidyl carrier
comprises SEQ ID NO:2 or SEQ ID NO:3.
13. The method of claim 11, wherein the MPG peptidyl carrier and
the antiviral peptide are in the form of a complex.
14. The method of claim 13, wherein the complex comprises the MPG
peptidyl carrier and the antiviral peptide at a ratio of about 20
molecules of the MPG peptidyl carrier for 1 molecule of the
antiviral peptide.
15. A method for treating or inhibiting an HIV infection,
comprising administering to a human in need thereof a
therapeutically effective amount of an inhibitor of HIV
replication, wherein: the inhibitor of HIV replication comprises a
chimeric peptide; and the chimeric peptide comprises: (a) a
decapeptide containing (from the N-terminus to the C-terminus) a
basic amino acid at position 1; an acidic amino acid at positions 2
and 5; a tryptophan at positions 4, 7, and 8; a threonine,
isoleucine or valine at position 3; a threonine, alanine, or
glutamine at position 6; a threonine, alanine, valine, isoleucine,
methionine, or aspartate at position 9; and a glutamate, aspartate
or asparagine at position 10; wherein the decapeptide inhibits the
dimerization of HIV reverse transcriptase, and (b) an MPG peptidyl
carrier peptide.
16. The method of claim 15, wherein the basic amino acid at
position 1 is lysine or arginine.
17. The method of claim 15, wherein the acidic amino acid at
position 2 is glutamate.
18. The method of claim 15, wherein the amino acid at position 5 is
glutamate.
19. The method of claim 15, wherein the inhibitor of HIV
replication further comprises a pharmaceutically acceptable
excipient.
20. The method of claim 15, wherein the MPG peptidyl carrier
peptide is SEQ ID NO:2 or SEQ ID NO:3.
21. The method of claim 15, wherein the decapeptide is selected
from the group consisting of KETWETWWTE (SEQ ID NO:1), KETWATWWTE
(SEQ ID NO:22), and KEAWETWWTE (SEQ ID NO:23)
22. The method of claim 15, wherein the chimeric peptide is SEQ ID
NO:4.
23. The method of claim 15, wherein the chimeric peptide is SEQ ID
NO:6.
24. The method of claim 1, wherein the method further comprises
administering one or more additional anti-HIV medicaments.
25. The method of claim 15, wherein the method further comprises
administering one or more additional anti-HIV medicaments.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Divisional Application of U.S. patent
application Ser. No. 09/648,557, filed Aug. 25, 2000, now U.S. Pat.
No. ______, the specification of which is incorporated herein by
reference in its entirety for all purposes.
FIELD OF THE INVENTION
[0002] The invention is drawn to a novel class of drugs directed
against HIV, inhibiting the dimerization of reverse transcriptase
of the virus, and to a method of treatment of HIV infections, in
particular multidrug-resistant HIV infections.
BACKGROUND OF THE INVENTION
[0003] In the last decade, a number of molecules have become
available for the treatment of HIV-infected individuals.
Therapeutic regimens based on the combination of reverse
transcriptase inhibitors and protease inhibitors have been shown to
reduce plasma HIV-1 RNA to undetectable levels in patients,
increase CD4 cell counts and delay progression toward AIDS.
[0004] HIV reverse transcriptase (RT) inhibitors that target the
polymerase activity of RT, can be subdivided into two classes of
potent agents: nucleosides that terminate viral DNA synthesis, such
as zidovudine (AZT), dideoxyinosine (ddI) and dideoxycytidine
(ddC), and nonnucleoside analogs that bind to a hydrophobic cavity
adjacent to the polymerase active site such as nevapirine (1).
However, these agents present several limitations, including
toxicity which sometimes requires patient's treatment to be
suspended (2), and the emergence of resistant strains which are
generated through the exceptionally high rate of mutagenesis of RNA
viruses (3-6). For example, resistance to zidovudine is conferred
by amino acid changes that appear in an orderly fashion: a K70R
mutation first, followed by T125F/Y, M4IL, D67N, and K219Q
mutations (7,8). Similarly, other mutations correlate with
resistant phenotype to other RT inhibitors (9). Thus, the
development of novel compounds that are active against
multidrug-resistant HIV variants is urgently needed.
[0005] An interesting feature of HIV-1 RT is that the dimeric form
of the enzyme consisting of two polypeptides p66 and p51, is
absolutely required for its catalytic activities (10). Based on the
x-ray crystallographic structure of HIV-1 RT, it was previously
demonstrated that the first interaction between p66 and p51 occurs
in a Tryptophan (Trp)-rich hydrophobic cluster located in the
connection subdomain of the two subunits and is followed by a
conformational change involving the thumb and the finger subdomains
of p51 as well as the RNase-H and the palm subdomains of p66
(11).
SUMMARY OF THE INVENTION
[0006] The present invention is based on the concept that the
dimerization process of RT could be an interesting target for AIDS
chemotherapy, and on the description of new inhibitors of HIV
replication, based on the inhibition of RT dimerization. These
inhibitors comprise peptides that will interact with the conserved
motif necessary for dimerization of the p51 and p66 subunit of the
HIV-RT.
[0007] Based on the concept that a small ligand of the connection
subdomains could inhibit RT dimerization, a short 10-residue
synthetic peptide (p7) derived from the Tip-rich cluster at the
interface of the connection subdomains of the p66 and p51
(KETWETWWTE; residues 395-404 of HIV-1 BH.sub.10 RT, SEQ ID NO:1)
was designed. This peptide p7 is a powerful inhibitor of HIV-1 RI
dimerization in vitro and abolishes the production of viral
particles in HIV-1 BRU-infected cultured CEM cells at a
concentration of 10.sup.-7 M, or 10.sup.-8 M when complexed with
the carrier peptidyl system MPG previously shown to improves the
delivery of molecules into cells (14 and 15, both incorporated
herein by reference in their totality). Interestingly, p7 does not
exhibit any toxicity in CEM cells at concentrations below 10.sup.-5
M. These encouraging studies prompted to pursue the
characterization of this compound as a model for potential new
antiviral drugs.
[0008] The present application demonstrates the potency of the
MPG/p7 complex in the abolition of the production of HIV-1 and
HIV-2 viruses and demonstrates that MPG/p7 is also a potent
inhibitor of drug resistant adapted HIV-1 strains.
DESCRIPTION OF THE FIGURES
[0009] FIG. 1: Effect of different concentrations of MPG/p7 (SEQ ID
NO:1) on HIV-1 and HIV-2 in CEM cell cultures. CEM cells exposed to
100 .mu.l of viral suspensions containing 1000.times.50% tissue
culture infective dose (TCID.sub.50)/ml of HIV-1 BRU (left panels)
or HIV-2 ROD (right panels). a, HIV-infected cells were cultured in
medium alone (white diamonds) or medium supplemented with MPG/p7 at
10.sup.-7 M (black circles), 10.sup.-8M, (black up triangles),
10.sup.-9 M (black diamonds), and 10.sup.-10 M (black squares). b,
As controls, HIV-infected cells were treated with either
azidothymidine (AZT: 10.sup.-5 M) (white squares) or peptide
MPG/p237 (SEQ ID NO:5) at 10.sup.-6 M (black down triangles). Viral
production was monitored by measuring RT activity twice a week post
infection. Culture supernatants from virus-free CEM were tested as
a control (white circles, FIG. 1b).
[0010] FIG. 2: Effect of MPG/p7 (SEQ ID NO:1) on replication of
different subtypes of HIV-1 and HIV-2 studied using MAGIC5 cells.
MAGIC-5 cells were incubated with 50 .mu.l of stock HIV preparation
corresponding to 1000.times.TCID.sub.50/ml. a, HIV-1 BRU; b, HIV-1
RF; c, HIV-1 SF2 (FIG. 2.A); d, HIV-I NDK; e, HIV-I ELI; f, HIV-2
ROD; g, HIV-2 EHO (FIG. 2.B) in medium alone (lane 2), medium
supplemented with AZT (10.sup.-5 M) (lane 3), MPG/p7 at 10.sup.-7
and 10.sup.-8 M (lane 4 and 5 respectively), or MPG/p237 (SEQ ID
NO:5) at 10.sup.-6 M (lane 6) additive. After 3 days in culture,
13-gal activity was evaluated in cell lysates by measuring
absorbance at 410 nm. .beta.-gal activity in uninfected MAGIC5
cells was measured as control (lane 1). All results have been
normalized with respect to .beta.-gal activity induced by each
virus (100% induction). The calculated values represent means of
duplicate. Each figure is representative of at least three
independent experiments.
[0011] FIG. 3: Effect of MPG/p7 (SEQ ID NO:1) on replication of
reference escape variant viruses. MAGIC-5 cells were incubated with
50 .mu.l of stock HIV preparation corresponding to
1000.times.TCID.sub.50/ml of a, HIV-1 BRU; b, HIV-I RTMF; c, HIV-1
RTMC; d, HIV-1 74V (FIG. 3.A); e, HIV-1 N119; f, HIV-1 RTMDR1 (FIG.
3.B), in medium alone (lane 2) or medium supplemented with AZT at
10.sup.-5 M, 10.sup.-6 M, 10.sup.-7 M, 10.sup.-8 M, 10.sup.-9 M,
and 10.sup.-10 M (lanes 3 to 8 respectively), MPG/p7 at 10.sup.-5
and 10.sup.-7 M (lanes 9 and 10), or MPG/p237 at 10.sup.-6 M (lane
11) additive. .beta.-gal activity was evaluated on day 3 after
virus exposure. .beta.-gal activity in uninfected MAGIC5 cells was
measured as control (lane 1). All results have been normalized (see
legend of FIG. 2). The calculated values represent means of
duplicate. Each figure is representative of at least three
independent experiments.
[0012] FIG. 4: Effect of MPG/p7 (SEQ ID NO:1), peptide p7+ (SEQ ID
NO:6) and peptide p7++ (SEQ ID NO:7) on replication of reference
viruses. MAGIC-5 cells were incubated with 50 .mu.l of stock HIV
preparation corresponding to 1000.times.TCID.sub.50/ml of a, HIV-1
BRU; b, HIV-2 ROD (FIG. 4.A); c, HIV-1 RTMF; d, HIV-1 RTMC (FIG. 4.
B); e, HIV-I 74V; f, HIV-1 RTMDR1 (FIG. 4.C). NI: non infected;
BRU: infected by virus BRU without inhibitor (or other viruses in
other panels); AZT: medium supplemented with AZT at 10.sup.-5 M;
MPG/p7: medium supplemented with MPG/p7 at the indicated
concentration; p7+: medium supplemented with p7+ at the indicated
concentration; p7++: medium supplemented with p7++ at the indicated
concentration; 237+: medium supplemented with MPG/237 (SEQ ID NO:5)
at the indicated concentration; .beta.-gal activity was evaluated
on day 3 after virus exposure. All results have been normalized
(see legend of FIG. 2). The calculated values represent means of
duplicate. Each figure is representative of at least three
independent experiments.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0013] The present invention is therefore drawn to an inhibitor of
HIV replication, comprising a peptide or analog comprising a
decapeptide, said decapeptide containing (from the N-terminus to
the C-terminus) a basic amino acid in position 1, an acidic amino
acid in positions 2 and 5, and a tryptophan in positions 4, 7, and
8.
[0014] By inhibition of HIV replication, it is meant an inhibition
of the production of viral particles from infected cells. This
inhibition may be measured by different means, in particular as
described in Morris et al. (15, which is incorporated herein by
reference in totality).
[0015] In particular, an inhibitor of HIV replication according to
the present invention reduces the .beta.-galactosidase activity
measured from MAGIC-5 cells by at least 50%, more preferably 70%,
the most preferably 90% in the conditions reported in the examples
of the present invention.
[0016] In a preferred embodiment, the basic amino acid in position
1 is arginine, or more preferably lysine.
[0017] In another embodiment, the acidic amino acid in position 2
is glutamate (also named glutamic acid).
[0018] In another embodiment, the acidic amino acid in position 5
is glutamate.
[0019] In another embodiment, the amino acid in position 3 is
chosen in the group consisting of threonine, isoleucine and valine,
and is preferably threonine.
[0020] In another embodiment, the amino acid in position 6 is
chosen in the group consisting of threonine, alanine and glutamine
and is preferably threonine or alanine.
[0021] In another embodiment, the amino acid in position 9 is
chosen in the group consisting of threonine, alanine, valine,
isoleucine, methionine, and aspartate (also named aspartic acid),
and is preferably threonine.
[0022] In another embodiment, the amino acid in position 10 is
chosen in the group consisting of glutamate, aspartate and
asparagine, and is preferably glutamate or aspartate, more
preferably glutamate.
[0023] In a preferred embodiment, the inhibitor of the invention
comprises a decapeptide containing (from the N-terminus to the
C-terminus) a basic amino acid in position 1, an acidic amino acid
in positions 2 and 5, and a tryptophan in positions 4, 7, and
8.
[0024] In another embodiment, the inhibitor of the invention is a
decapeptide containing (from the N-terminus to the C-terminus) a
basic amino acid in position 1, an acidic amino acid in positions 2
and 5, and a tryptophan in positions 4, 7, and 8.
[0025] The peptide or analog that can be used preferably covers the
residues 389-407 of the HIV-RT, more preferably 395-404.
[0026] By peptide or analog, it is meant to understand a molecule
comprising a series of amino acids, that can be natural or not,
said molecule being linear or circular, and capable of being
modified, by linkages such as glycosylation or branches of amino
acids such as glutamylation on glutamate(s).
[0027] A peptide according to the present invention can be obtained
by genetic engineering, after introduction of a DNA vector carrying
a nucleic acid sequence coding for the peptide sequence into a host
cell (procaryotic or eucaryotic cell, such as bacteria, yeast,
mammalian cell), and producing the peptide within said host cell.
Some induction systems allow the production of a large amount of
peptide, and are techniques well known by the person skilled in the
art.
[0028] The peptides or analogs according to the present invention
can also be obtained by chemical synthesis, using one of the many
known peptidic synthesis. One could cite the techniques using solid
phases, total or partial, by fragment condensation, or a synthesis
in classical solution.
[0029] The peptides or analogs of the invention may comprise non
natural or modified amino acids. Among these non naturally
occurring amino acid, one could cite without being limitative,
ornithine, norleucine, norvaline, hydroxyproline, hydroxylysine,
ethylglycine, ethylasparagine. A list of modified amino acids has
been edited by the WIPO in the definition of the sequence norm ST
25.
[0030] The peptides or analogs according to the present invention
can contain modifications of the amino acids that are found
naturally or not, among them glycosylation.
[0031] The sequence of the peptides or analogs according to the
present invention can also be modified, without modification of the
biological activity (inhibition of HIV-RI replication, in
particular through inhibition of HIV-RT dimerization), in
particular to increase their solubility, preferably in aqueous
solvents.
[0032] The peptides or analogs according to the invention may be
modified in order to increase their stability in vitro and/or in
vivo. For example, one can use D amino acids and/or block the N
and/or C-termini of the peptides.
[0033] It is possible to replace some minor amino-acids to increase
the stability or allow a better penetration of the peptide or
analog in the cell.
[0034] It is to understand that the acceptable modifications to the
peptides or analogs maintain the biological activity of said
molecules, which is to inhibit HIV replication.
[0035] The process for the purification of peptides are known by
the person skilled in the art. Recombinant peptides can be purified
from lysates or cellular extracts, from the supernatant of the
culture medium by techniques used individually or in
combination.
[0036] The techniques that can be used to purify peptides prepared
through a recombinant host or by chemical synthesis include
fractionment, chromatography, immunoaffinity techniques, using
monoclonal or polyclonal specific antibodies.
[0037] The inhibitor according to the present invention exhibits a
better potency when it further comprises a vector allowing the
penetration of the peptide or analog into a mammalian cell.
[0038] The person skilled in the art can design such a vector,
which preferably is comprised in the group consisting of liposomes,
polymeric protein-binding cations, proteins, peptides, micro- or
nanoparticles.
[0039] In a preferred embodiment of the invention, the vector used
to facilitate the penetration of the peptide in the inhibitor of
the present invention comprises the peptide MPG (SEQ ID NO:2), the
amphipatic sequence of peptide MPG (SEQ ID NO:3) or an analog
thereof.
[0040] In a preferred embodiment of the invention, the vector used
to facilitate the penetration of the peptide in the inhibitor of
the present invention is the peptide MPG (SEQ ID NO:2), the
amphipatic sequence of peptide MPG (SEQ ID NO:3) or an analog
thereof.
[0041] In a preferred embodiment, the peptide and the vector in the
inhibitor according to the present invention are in the form of a
complex.
[0042] In another embodiment, the peptide and the vector in the
inhibitor according to the present invention are linked by a
covalent liaison.
[0043] In a very preferred embodiment, the inhibitor according to
the invention is formed by a peptide comprising peptide p7 (SEQ ID
NO:1) and peptide MPG (SEQ ID NO:2) or the amphipatic sequence of
peptide MPG (SEQ ID NO:3), or analogs thereof.
[0044] In the most preferred embodiment, the inhibitor according to
the invention is peptide p7++ (retroinhibase 1, SEQ ID NO:4), or an
analog thereof.
[0045] The invention is also drawn to a pharmaceutical composition
comprising an inhibitor of HIV replication according to the
invention, and an appropriate excipient. Said compositions are
preferably formulated for administration to mammals, in particular
human beings. They are preferably formulated to be administrated by
oral, sublingual, subcutaneous intramuscular, intravenous,
transdermal, rectal way.
[0046] The pharmaceutical composition may be a tablet, a capsule, a
powder, a pill, a suppository, a solution (injectable by a method
as previously cited) or a suspension.
[0047] The excipient may be gelatin, starch, lactose, arabic gum,
talc, or other known pharmaceutical vehicles. The tablets may be
coated by sucrose, or other appropriate compounds.
[0048] The pharmaceutical composition according to the invention
may be treated as to achieve a sustained or retarded activity, or
for the release of a predetermined amount of inhibitor in a
continuous way.
[0049] The capsule may be obtained by mixing the inhibitor with a
diluent and pouring the mixture in soft or hard capsules.
[0050] A syrup may be obtained by mixing the inhibitor with an
sweetener, an antiseptic, a tasting agent, and an appropriate
colorant.
[0051] Powders or granules may contain the inhibitor mixed with
dispersion agents, or wetting agents, optionally with tasting
agents and/or sweeteners.
[0052] For rectal administration, suppositories may be prepared
with binding agents, melting at rectal temperature, such as cocoa
butter or polyethyleneglycols.
[0053] For injectable administration, one could use aqueous
suspensions, saline isotonic solutions or sterile solutions that
contains dispersions agents, and/or wetting agents
pharmacologically compatibles.
[0054] The inhibitor may also be formulated as a microcapsule, with
possibly one or more additive supports.
[0055] The examples in the present application show that the
inhibitors of the invention are very potent against the replication
of HIV strains in vitro. Furthermore, they are also very potent
against both HIV-I and HIV-2 strains, as well as against drug- and
multidrug-resistant strains.
[0056] Therefore, the invention is also drawn to the use of an
inhibitor, or a composition according to the invention, for the
manufacture of a medicament to be used in the treatment of an HIV
infected patient, whether HIV is a HIV-1, HIV-2, drug sensitive,
drug-resistant or multidrug-resistant HIV virus.
[0057] It is foreseen that the medicament of the present invention
will be used simultaneously or in combination with one or more
other anti-HIV medicament(s). Indeed, the best current clinical
results for limiting HIV infections are obtained by using multiple
drugs at the same time. The invention presents a new therapeutic
class of molecules to be used against HIV, and shall therefore be
added to the current treatment regimens.
[0058] The other anti-HIV medicaments that can be used at the same
time as the medicament or the inhibitor of the invention include
protease inhibitors and inhibitors of the HIV-RT, such as
nucleoside or non-nucleosides inhibitors.
[0059] It is also worth noting that the inhibitors according to the
present invention are directed against conserved region of the
HIV-RT, that is essential for the dimerization of the protein.
Therefore, by using the inhibitor of the invention, one can prevent
the dimerization of the HIV-RT, which may prevent the reverse
transcription of the virus RNA to DNA, and its integration within
the genome.
[0060] Furthermore, the inhibitors and compositions of the
invention are advantageous in that they target a conserved region
of the virus genome, that is probably not very prone to mutations,
as it is essential for the dimerization of the HIV-RT protein. It
is therefore expected that there will be less resistant strains to
the inhibitors of the invention than with other inhibitors of
HIV-RT, such as nucleoside analogs.
[0061] The inhibitors of the present invention show an inhibition
of virus replication for concentrations in the range of 10.sup.-7
or 10.sup.-8 M in vitro, that is lower than the concentration
needed for AZT. Furthermore, cell toxicity is only observed for
inhibitor concentration 10,000 times higher.
[0062] It is therefore foreseen that the inhibitors or the
composition according to the present invention will be administered
at a dose that will allow them to be effective. Such a dose is said
to be therapeutically effective, i.e. anti-virally effective,
without a reduced toxicity.
[0063] According to the literature that reports on the use of
peptides as therapeutic agents (40), the medicament of the
invention may be administered at a dose of about 1 to 1000 mg/day,
or more preferably at a dose of about 20 to 700 mg/day.
[0064] The invention is also drawn to a method for treating or
inhibiting an HIV infection comprising administering to a human in
need thereof a therapeutically effective (anti-virally effective)
amount of an inhibitor, or a composition according to the
invention, optionally in combination with a therapeutically
effective amount of one or more other anti-HIV medicament(s) (such
as nucleosides or non-nucleosides inhibitors of the reverse
transcriptase, protease inhibitors).
[0065] The method is effective against HIV-I or HIV-2, and
particularly against drug- or multidrug-resistant HIV.
EXAMPLES
Example 1
Methods
[0066] 1.1 Viruses
[0067] The HIV strains used in this study were already described:
HIV-I BRU (28), HIV-2 ROD (29), HIV-I ELI (30), HIV-2EHO (31,32),
HIV-1 NDK (33), HIV-1 RF (34), HIV-1 SF2 (35), nevirapine-resistant
HIV-INI19 (20), HIV-1 RTMC (21), HIV-1 RTMF (18), HIV-1 74V (36),
and HIV-1 RTMDR1 (24). These viruses were propagated in CEM cells
(a CD4+/CXCR4+ human T-cell line).
[0068] 1.2. Cells
[0069] The CD4+, CXCR4+ lymphoblastoid CEM cell line was purchased
from the American Type Culture Collection (CCRF-CEM, ATCC # CCL
119, Catalogue of cell lines and hybridomas, ATCC, Bethesda, Md.).
CEM cells were cultured in RPMI 1640 medium containing 1%
penicillin-streptomycin (PS) antibiotic mixture, 1% glutamax
(Gibco-BRL, Eragny, France) and 10% FCS (Gibco), to a density of
5.times.10.sup.5 cells/ml in a 5% CO.sub.2 atmosphere. The
HeLa-LTR-.beta.gal indicator cell line (37) stably transfected with
CD4 and CCR5 (MAGIC-5) cells was previously described (38), were
grown in DMEM containing 1% PS, 1% glutamax, 1 mg/ml G418, and 10%
FCS.
[0070] 1.3. Peptides
[0071] Peptides (p7 (SEQ ID NO:1), p237 (SEQ ID NO:5), MPG (SEQ ID
NO:2), p7++ (SEQ ID NO:4), and p7+ (SEQ ID NO:6)) were synthesized
by solid phase peptide synthesis using
aminoethyldithio-2-isobutyric acid-expensin resin with a 9050
Pepsynthetizer (Millipore, UK) according to the
Fmoc(N-(9-fluorenyl)methoxycarbonyl)/tert-butyl method, purified by
semi-preparative HPLC and identified by electrospray mass
spectrometry and amino acid analysis. In some case, to increase
their stability, the peptides were acetylated at the N terminus and
linked to a cysteamide group at the C-terminal part as previously
described (14).
[0072] 1.4. Formation of p7/MPG Complex
[0073] Peptide p7 (SEQ ID NO:1) and MPG (SEQ ID NO:2) were mixed,
and peptide p7 binds to MPG (probably the hydrophobic domain), with
saturation taking place for a concentration of p7 about 20-fold
lower than of MPG. From the K.sub.d and the saturation
concentration, the ration was estimated to 30 molecules of MPG for
one molecule of p7. The MPG/p7 complex was further assessed as a
complex of p7-MPG at a 1/20 ratio (15, incorporated herein by
reference in its totality).
[0074] 1.5. Infection of Cells
[0075] RI activity assay. CEM cells were incubated for 30 mm at
4.degree. C. with 100 .mu.l of stock HIV preparation corresponding
to 1000.times.50% tissue culture infective dose (TCID.sub.50)/ml,
then cells were washed five times and cultured at 5.times.10.sup.5
cell/ml in 24-well microplates in the presence or absence of MPG/p7
(at 10.sup.-8 and 10.sup.-7 M) or AZT (AZT was purchased from
Boehringer Mannheim, Germany) additive. Viral production was
monitored twice a week by measuring reverse transcriptase activity
in 1 ml of cell-free supernatant as previously described (39).
[0076] .beta.-gal activity assay. MAGIC-5 cells expressing the
.beta.-ga1 reporter gene cloned downstream of the HIV-1 LTR
promoter were plated in 24-well plates at 5.times.10.sup.-5
cells/ml and incubated with 50 .mu.l of stock HIV preparation
corresponding to 1000.times.50% tissue culture infective dose
(TCID.sub.50)/ml in the presence or absence of MPG/p7 (at 10.sup.-5
and 10.sup.-7 M) or AZT additive. After 3 days in culture, cells
were lysed and .beta.-gal activity was determined by incubating 200
.mu.l of total cellular extracts for 1 h at 37.degree. C. in 1.5 ml
buffer containing 80 mM NA.sub.2HPO.sub.4, 10 mM MgCl.sub.2, 1 mM 2
ME and 6 mM o-nitrophenyl .beta.-D-galactopyranoside (ONPG).
.beta.-gal activity was evaluated by measuring absorbance at 410
nm.
Example 2
Inhibition of HIV-1 and HIV-2 Isolates Replication in CEM Cells by
MIPG/p7 Complex
[0077] MPG/p7 was previously shown to inhibit HIV-1 BRU RT
dimerization in vitro and HIV-1BRU replication in CEM cell culture
(15, incorporated herein by reference). The susceptibility of two
reference strains of laboratory-adapted HIV-1 and HIV-2 to MPG/p7
was first determined.
[0078] A representative experiment of inhibition of virus
production (HIV-1 BRU and HIV-2ROD) in infected CEM cells treated
with MPG/p7 (10.sup.-6 M), a 15-mer control peptide 237 (10.sup.-6
M) (SEQ ID NO:5), or AZT (10.sup.-5 M), is shown in FIG. 1. RT
activity monitored in cell free culture supernatant of infected
cells from day 3 to day 17 after virus exposure, indicated that
MPG/p7 at concentrations above 10.sup.-5 M, totally inhibits
HIV-1BRU replication during the 17 days of incubation whereas RT
activity observed at the end of culture in samples treated at
concentration of 10.sup.-9M revealed a very slow virus propagation
that was undetected at earlier time-points. Only a 3-days delay in
HIV-1 replication was found at a concentration of 10.sup.-10 M of
MPG/p7.
[0079] Under similar experimental conditions HIV-2ROD propagation
was strongly delayed at concentrations of MPG/p7 above 10.sup.-7 M,
whereas a 3-days delay in HIV-2 replication was observed using a
concentration of 10.sup.-9 M of MPG/p7.
[0080] This result was confirmed by monitoring the expression of
HIV-1 antigen by p24.sup.gag antigen capture assay on day 17 post
infection (data not shown).
Example 3
Inhibition of HIV-1 and HIV-2 Isolates Replication in MAGIC5 Cells
by MPG/p7 Complex
[0081] The pattern of reactivity of MPG/p7 using several isolates
from different clades of HIV-1 and HIV-2 isolates (see Table 1) was
next determined.
[0082] The efficiency of MPG/p7 monitored using the previously
described MAGIC5 transfectant cells that express surface CD4, CXCR4
and CCR5 receptors and contain a reporter gene under control of an
HIV-1 promoter that can be induced upon infection of the cells.
This assay was chosen because it gives a result within 72 h and
requires much less peptide in each experiment than classical
infection assays.
[0083] All strains tested were found to be susceptible to MPG/p7 at
concentrations of 10.sup.-8 M (FIG. 2). This included X4 and R5/X4
strains of HIV-1 from clades B and D and HIV-2 clades A and B.
[0084] Although only HIV-1 clades B and D and HIV-2 clades A and
.beta. isolates were tested, it can be assumed that the 7 isolates
that were used in the present study are representative of the
different sequences that can be encountered within HIV-1 clades A,
B, C, D, F, G, H, O and HIV-2 clades A and B.
[0085] Indeed, comparisons of the sequence of residues 395-404 of
HIV-1 BH.sub.10 RT (KETWETWWTE, SEQ ID NO:1) to the corresponding
sequences available from the Los Alamos data base (9) (Table 1)
reveals that the major substitutions observed between the consensus
B and the other clades of HIV-1 are found in one of the HIV-1 B or
D, or HIV-2 viruses tested.
[0086] For example, the HIV-1 clade C (see consensus C sequence)
that is most predominant in India and causes more than 70% of
infections in southern Africa and 96% in northern Africa shows a
T.sub.404A substitution also encountered in HIV-1 RF and HIV-1 NDK
and an E.sub.404D substitution also encountered in HIV-2 EHO.
[0087] Therefore, in most cases substitutions do not alter the
character of the residues, confirming that this region is highly
conserved in HIVs.
[0088] These observations suggest that irrespectively of type,
clade and geographic origin, all human lentiviruses containing a
decapeptide containing (from the N-terminus to the C-terminus) a
basic amino acid in position 1, an acidic amino acid in positions 2
and 5, and a tryptophan in positions 4, 7, and 8 at the interface
of the connecting subdomains of the p66 and p51 subunits can
potentially be inhibited by MPG/p7.
Example 4
Crystallographic Analysis of the HIV-RT
[0089] The crystallographic structure of HIV-1 RT, reveals that the
residues 395-404 are involved in the p66/p51 interface contacts and
are essential in the stabilization of both the connection
subdomain.
[0090] Secondary structure predictions and molecular modeling
suggest that in all the isolates these residues are folded into an
.alpha.-helix, as observed in the X-ray structure of HIV-1
(16,17).
[0091] In both subunits, the highly conserved Trp residues,
Trp.sub.398, Trp.sub.401 and Trp.sub.402 form a cluster of aromatic
residues together with Tyr.sub.405, Trp.sub.410, and Phe.sub.416
which stabilizes the dimer interface by intra- and inter-subunit
contacts. In p51 the hydrophobic cluster involves other contacts
which maintains the conformation of the palm domain of p51
(Trp.sub.24, Phe.sub.61, Leu.sub.368, Leu.sub.391, Val.sub.372) and
the thumb-domain of p66 (Arg.sub.356, Arg.sub.358, Gln.sub.373). In
p66, additional contacts are made with the RNase-H domain
(Va1.sub.423, Leu.sub.425).
[0092] Analysis of the amino acid substitutions indicated that
whatever the sequence mentioned in Table 1, the property of the
residues is conserved in order to maintain the organization of the
hydrophobic pocket and the .alpha.-helix conformation.
[0093] Substitution K.sub.395R or E.sub.399D) retains the basic
characteristic, essential for helix stability and interactions with
Trp.sub.24, Phe.sub.416 and Trp.sub.414.
[0094] Substitution E.sub.396D keep the acidic property essential
for the interaction with residues Gln.sub.394 in p51 and the two
Arg.sub.356 and Arg.sub.358 in p66.
[0095] Thr.sub.397 is conserved in all of the consensus sequences,
excepted in HIV-2 consensus A, in both subunits this residue is
surrounded by hydrophobic residues, which cannot be altered when
replaced by isoleucine or valine.
[0096] Thr.sub.400 is one of the most variable residue of this
motif, therefore none of the substitution alters the organization
of the aromatic cluster.
[0097] Moreover, the substitution T.sub.400Q observed in all HIV-2
consensus increases interaction between p51 and p66 subunits and
may explain the higher stability of HIV-2 RT.sup.11.
[0098] Taken together, these data indicate that MPG/p7 inhibits a
wide range of HIVs. Based on these results we conclude that the
integrity of the aromatic cluster which is essential for dimer
formation as well as for the structural integrity of both subunits,
is conserved in all of the isolates described in Table 1. This
explains why a drug like p7, which targets the aromatic cluster
prevents the dimer formation of all of these isolates.
Example 5
Susceptibility of Anti-RT Drug Resistant HIV-1 Strains to
MPG/p7
[0099] The phenotypic identification of drug-resistant HIV-1
emerging during unsuccessful antiretroviral therapy has enable the
definition of drug resistant genotypes of HIV-1.
[0100] Several mutations in RT are consistently in association with
resistance to one or more anti-RT drugs. The growing number of
reports documenting mutations which confer resistance to both
nucleoside and non-nucleoside RT inhibitors indicates that one of
the first selection criteria that a new antiviral compound
targeting RT should meet, is its capacity to inhibit anti-RT drug
resistant HIV-1 strains.
[0101] According to the compilation of mutations in HIV RT
published by the Los Alamos National Laboratory (9), there are at
least 45 amino acid residues in RT for which mutations result in a
significant change in the virus susceptibility to one or more
anti-RT drugs.
[0102] Interestingly, and to the best of our knowledge, there is
actually no mutation reported affecting the residues of HIV-1 RT
that are target for MPG/p7. Moreover, that mutation of Trp.sub.398
and Trp.sub.410 (HIV-1 BH10) strongly affected the stability of the
dimeric form of HIV-1 RT in vitro (Morris and Divita, unpublished
data), strengthens the hypothesis that mutations occurring in this
region may affect RT dimerization thereby disabling viral
replication.
[0103] To determine whether mutant HIV-1s that resist to both
nucleoside and/or nonnucleoside RT inhibitors are sensitive to
MPG/p7, five reference strains of anti-RT drug resistant HIV-1 were
assayed for susceptibility to MPG/p7.
[0104] Table 2 summarizes the characteristics of RT drug resistant
phenotype of these viruses, namely HIV-1 RTMF, HIV-1 RTMC, HIV-1
74V, HIV-1 N119 and HIV-1 RTMDR1, and the type of mutation
conferring these phenotypes.
[0105] The different escape mutant viruses studied turned to be
sensitive to MPG/p7 treatment (FIG. 3). It is however worth noting
that the concentration of MPG/p7 required to inhibit HIV-1 RTMC and
HIV-I RTMDR1 (FIGS. 3c and f) was higher than that required to
block the other viruses. For example, 10.sup.-6 M of MPG/p7 was
required for completely inhibit HIV-1 RTMC (data not shown).
[0106] The fact that the sensitivity to zidovudine of HIV-1 RTMC
and HIV-1 RTMF escape mutant strains was very similar in the
present experiment and in the experiment previously reported by
Larder and co-workers (18), validated the observations relatively
to MPG/p7 effect on the different escape mutant viruses.
Example 6
Inhibition of HIV Replication by Peptide D.sup.7++
[0107] In order to further study the properties of peptide p7,
chimeric peptides having the transmembranaire transport properties
of MPG (amhipathic sequence) and anti HIV-RT properties of p7 were
designed and synthesized.
[0108] Peptide p7++ (SEQ ID NO:4) and p7+ (SEQ ID NO:6) were used
on MAGIC-5 cells, infected with the ROD (HIV-2) and BRU (HIV-1)
strains.
[0109] FIG. 4 shows that peptide p7++ (retroinhibase 1) exhibits
the same activity than the MPG-p7 complex at about the same
concentration.
[0110] The use of peptide p7++ on cells infected by drug-resistant
viruses demonstrates that this peptide is also capable to inhibit
the replication of such strains (FIG. 4).
TABLE-US-00001 TABLE 1 Amino acid alignment with the p7 sequence of
BH10 strain Country SEQ of ID Type Strain Clade origin Sequence NO
HIV-1 395 404 BH10 KETWETWWTE 1 BRU B France KETWETWWTE 1 RF B
Haiti/ KETWEAWWTE 7 USA SF2 B USA KETWEAWWME 8 NDK D Zaire
KETWETWWIE 9 ELI D Zaire KETWETWWAE 10 HIV-2 ROD A Cape REIWEQWWDN
11 EHO B Ivory RETWDQWWTD 12 Coast HIV-1 395 (p7) 404 KETWETWWTE 1
Consensus A KETWET/AWWTE/D 13 Consensus B KETWET/AWWME 14 Consensus
C KETWEAWWTD 15 Consensus D KETWET/AWWXE/D 16 (X = T/A/V/I)
Consensus F KETWDTWWTE 17 Consensus G KETWEVWWTE 18 Consensus H
KETWETWWTE 1 Consensus O RETWETWWAD 19 HIV-2 Consensus A
RE/DZWEQWWDN/D 20 (Z = T/V/I) Consensus B RETWDQWWTD 21
Sequences are from ref. 9
TABLE-US-00002 TABLE 2 Main characteristics of anti-RT drug
resistant HIV-1 isolates Strain RT Genotyping Phenotype HIV-1 RTMF
215Y AZT-resistant HIV-1 RTMC 67N, 70R, 215F, 219Q AZT-resistant
HIV-1 74V 74V resistant ddI and ddC HIV-1 N119 181C resistant to
nevapirine and non nucleoside RT inhibitors HIV-1 RTMDR1 41L, 74V,
106A, 215Y resistant to AZT, ddI, nevapirine, non nucleoside RT
inhibitors
Viruses phenotype and reverse transcriptase genotype are adapted
from refs. 18, 20, 21, 23, and 36. HIV-1 RTMF, RTMC, 74V, RTMDR1,
and N119 viruses utilize CXCR4 (some isolates are R5X4 dual tropic
strains).
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Sequence CWU 1
1
23110PRTHIVResidues 395-404 of HIV-1 BH10RT 1Lys Glu Thr Trp Glu
Thr Trp Trp Thr Glu1 5 10227PRTArtificial SequencePeptide MPG 2Gly
Ala Leu Phe Leu Gly Phe Leu Gly Ala Ala Gly Ser Thr Met Gly1 5 10
15Ala Trp Ser Gln Pro Lys Ser Lys Arg Lys Val 20 2536PRTArtificial
SequenceAmphipatic sequence of peptide MPG 3Gly Phe Leu Gly Ala
Ala1 5425PRTArtificial SequenceRetroinhibase 1, peptide p7++ 4Gly
Phe Leu Gly Ala Ala Gly Ser Thr Met Gly Ala Trp Ser Gln Lys1 5 10
15Glu Thr Trp Glu Thr Trp Trp Thr Glu 20 25515PRTHIVPeptide p237
5Arg Gly Thr Lys Ala Leu Thr Glu Val Ile Pro Leu Thr Glu Asp1 5 10
15621PRTArtificial SequencePeptide p7+ 6Gly Ala Leu Phe Leu Gly Phe
Leu Gly Ala Ala Lys Glu Thr Trp Glu1 5 10 15Thr Trp Trp Thr Glu
20710PRTHIV-1 7Lys Glu Thr Trp Glu Ala Trp Trp Thr Glu1 5
10810PRTHIV-1 8Lys Glu Thr Trp Glu Ala Trp Trp Met Glu1 5
10910PRTHIV-1 9Lys Glu Thr Trp Glu Thr Trp Trp Ile Glu1 5
101010PRTHIV-1 10Lys Glu Thr Trp Glu Thr Trp Trp Ala Glu1 5
101110PRTHIV-2 11Arg Glu Ile Trp Glu Gln Trp Trp Asp Asn1 5
101210PRTHIV-2 12Arg Glu Thr Trp Asp Gln Trp Trp Thr Asp1 5
101310PRTHIV-1VARIANT6Xaa = Thr or Ala 13Lys Glu Thr Trp Glu Xaa
Trp Trp Thr Xaa1 5 101410PRTHIV-1VARIANT6Xaa = Thr or Ala 14Lys Glu
Thr Trp Glu Xaa Trp Trp Met Glu1 5 101510PRTHIV-1 15Lys Glu Thr Trp
Glu Ala Trp Trp Thr Asp1 5 101610PRTHIV-1VARIANT6Xaa = Thr or Ala
16Lys Glu Thr Trp Glu Xaa Trp Trp Xaa Xaa1 5 101710PRTHIV-1 17Lys
Glu Thr Trp Asp Thr Trp Trp Thr Glu1 5 101810PRTHIV-1 18Lys Glu Thr
Trp Glu Val Trp Trp Thr Glu1 5 101910PRTHIV-1 19Arg Glu Thr Trp Glu
Thr Trp Trp Ala Asp1 5 102010PRTHIV-2VARIANT2Xaa = Glu or Asp 20Arg
Xaa Xaa Trp Glu Gln Trp Trp Asp Xaa1 5 102110PRTArtificial
SequenceHIV-1 21Arg Glu Thr Trp Asp Gln Trp Trp Thr Asp1 5
102210PRTArtificial SequenceHomo Sapiens 22Lys Glu Thr Trp Ala Thr
Trp Trp Thr Cys1 5 102310PRTArtificial SequenceHomo Sapiens 23Lys
Glu Ala Trp Glu Thr Trp Trp Thr Glu1 5 10
* * * * *